The proposed study is based upon the successful synthesis of the first specific, potent multisubstrate analog inhibitor of the enzyme thymidylate synthetase. The prototype inhibitor consists of a 5,6,7,8-tetrahydro-8-deazafolate linked at N5 through a methylene bridge to C5 of 2'-deoxyuridylate. The inhibitor will be modified chemically in specific ways designed to (a) explore the active site of thymidylate synthetase through modification of the p-aminobenzoyl glutamate side chain (b) reduce the charge and increase the lipohilicity of these highly polar molecules such that entry to intact cells and subsequent conversion to active inhibitor may occur and (c) develop a "second generation" of flexible analogs expected to have still greater potency and specificity for the thymidylate synthetase. This approach should provide not only biochemical probes for elucidating the effects of specific inhibition of this key enzyme, but may also provide useful antitumor and, perhaps, antiviral agents.